拮抗菌TG26的鉴定及其抗菌蛋白BI的纯化和部分特性

从丝瓜根部分离出来的桔抗菌ＴＧ２６,根据形态特征和生理生化特性,鉴定为枯草芽抱杆菌（Ｂａｃｉｌｌｕｓｓｕｂｔｉｌｉｓ）。该菌株能分泌大量的抗菌蛋白,经ＳｅｐｈａｄｅｘＧ－１５０柱和ＦＰＬＣＭｏｎｏＱ柱层析后得到单一组分的抗菌蛋白,命名为ＢＩ。ＢＩ能抑制多种植物病原菌的生长,对热稳定,对蛋白酶部分敏感。经ＳＤＳ－ＰＡＧＥ和等电聚焦电泳测定其分子量约１４．５ｋＤ,等电点为５．５８。氨基酸组成分析表明,该蛋白宫含谷氨酸、酪氨酸和脯氨酸;并测定了Ｎ末端氨基酸的部分序列。     

Functional Identification of Conserved Residues Involved in Lactobacillus rhamnosus Strain GG Sortase Specificity and Pilus Biogenesis

In Gram-positive bacteria, sortase-dependent pili mediate the adhesion of bacteria to host epithelial cells and play a pivotal role in colonization, host signaling, and biofilm formation. Lactobacillus rhamnosus strain GG, a well known probiotic bacterium, also displays on its cell surface mucus-binding pilus structures, along with other LPXTG surface proteins, which are processed by sortases upon specific recognition of a highly conserved LPXTG motif. Bioinformatic analysis of all predicted LPXTG proteins encoded by the L. rhamnosus GG genome revealed a remarkable conservation of glycine residues juxtaposed to the canonical LPXTG motif. Here, we investigated and defined the role of this so-called triple glycine (TG) motif in determining sortase specificity during the pilus assembly and anchoring. Mutagenesis of the TG motif resulted in a lack or an alteration of the L. rhamnosus GG pilus structures, indicating that the TG motif is critical in pilus assembly and that they govern the pilin-specific and housekeeping sortase specificity. This allowed us to propose a regulatory model of the L. rhamnosus GG pilus biogenesis. Remarkably, the TG motif was identified in multiple pilus gene clusters of other Gram-positive bacteria, suggesting that similar signaling mechanisms occur in other, mainly pathogenic, species.     

Detection of transglutaminase activity using click chemistry

Transglutaminase 2 (TG2) is a Ca(2+)-dependent enzyme able to catalyze the formation of ε(γ-glutamyl)-lysine crosslinks between polypeptides, resulting in high molecular mass multimers. We have developed a bioorthogonal chemical method for the labeling of TG2 glutamine-donor proteins. As amine-donor substrates we used a set of azide- and alkyne-containing primary alkylamines that allow, after being crosslinked to glutamine-donor proteins, specific labeling of these proteins via the azide-alkyne cycloaddition. We demonstrate that these azide- and alkyne-functionalized TG2 substrates are cell permeable and suitable for specific labeling of TG2 glutamine-donor substrates in HeLa and Movas cells. Both the Cu(I)-catalyzed and strain promoted azide-alkyne cycloaddition proved applicable for subsequent derivatization of the TG2 substrate proteins with the desired probe. This new method for labeling TG2 substrate proteins introduces flexibility in the detection and/or purification of crosslinked proteins, allowing differential labeling of cellular proteins.     

Disruption of single-copy genes encoding acidic ribosomal proteins in Saccharomyces cerevisiae.

Using the cloned genes coding for the ribosomal acidic proteins L44 and L45, constructions were made which deleted part of the coding sequence and inserted a DNA fragment at that site carrying either the URA3 or HIS3 gene. By gene disruption techniques with linearized DNA from these constructions, strains of Saccharomyces cerevisiae were obtained which lacked a functional gene for either protein L44 or protein L45. The disrupted genes in the transformants were characterized by Southern blots. The absence of the proteins was verified by electrofocusing and immunological techniques, but a compensating increase of the other acidic ribosomal proteins was not detected. The mutant lacking L44 grew at a rate identical to the parental strain in complex as well as in minimal medium. The L45-disrupted strain also grew well in both media but at a slower rate than the parental culture. A diploid strain was obtained by crossing both transformants, and by tetrad analysis it was shown that the double transformant lacking both genes is not viable. These results indicated that proteins L44 and L45 are independently dispensable for cell growth and that the ribosome is functional in the absence of either of them.     

Saccharomyces cerevisiae ribosomal protein L26 is not essential for ribosome assembly and function

Ribosomal proteins play important roles in ribosome biogenesis and function. Here, we study the evolutionarily conserved L26 in Saccharomyces cerevisiae, which assembles into pre-60S ribosomal particles in the nucle(ol)us. Yeast L26 is one of the many ribosomal proteins encoded by two functional genes. We have disrupted both genes; surprisingly, the growth of the resulting rpl26 null mutant is apparently identical to that of the isogenic wild-type strain. The absence of L26 minimally alters 60S ribosomal subunit biogenesis. Polysome analysis revealed the appearance of half-mers. Analysis of pre-rRNA processing indicated that L26 is mainly required to optimize 27S pre-rRNA maturation, without which the release of pre-60S particles from the nucle(ol)us is partially impaired. Ribosomes lacking L26 exhibit differential reactivity to dimethylsulfate in domain I of 25S/5.8S rRNAs but apparently are able to support translation in vivo with wild-type accuracy. The bacterial homologue of yeast L26, L24, is a primary rRNA binding protein required for 50S ribosomal subunit assembly in vitro and in vivo. Our results underscore potential differences between prokaryotic and eukaryotic ribosome assembly. We discuss the reasons why yeast L26 plays such an apparently nonessential role in the cell.     

保加利亚乳杆菌H+-ATPase缺陷型菌株的筛选

【目的】从传统乳制品中筛选具有新霉素抗性的H+-ATPase缺陷的德氏乳杆菌保加利亚亚种自发突变株,为最终开发弱后酸化的酸奶发酵剂奠定基础。【方法】利用API 50 CH细菌鉴定系统和16s rRNA基因序列分析对菌株进行鉴定。新霉素作为筛选压力,筛选具有新霉素抗性自发突变菌株,比较亲本和突变菌株的H+-ATPase活力及其代谢情况。【结果】从内蒙古地区的传统发酵酸奶中分离鉴定出一株德氏乳杆菌保加利亚亚种（Lactobacillus delbrueckii subsp. bulgaricus）,并命名为KLDS 1.9201。以此为出发菌株,筛选出两株H+-ATPase缺陷的自发突变株,分别命名为KLDS 1.9201-1、KLDS 1.9201-4,它们的H+-ATPase活力分别比亲本KLDS 1.9201降低了46%和60%。在MRS培养基中生长24 h后,KLDS 1.9201、KLDS 1.9201-1和KLDS 1.9201-4对初始葡萄糖的代谢率分别为65%、41%和31%,终产物中乳酸的浓度分别为26g/L、18g/L和15g/L,突变菌株的生物量均低于亲本。【结论】H+-ATPase活力降低的德氏乳杆菌保加利亚亚种的自发突变株具有较低的生长速率和弱产酸能力,它们可被用于制作弱后酸化的酸奶发酵剂。     

Stepwise dissociation of yeast 60S ribosomal subunits by LiCl and identification of L25 as a primary 26S rRNA binding protein

Treatment of yeast 60S ribosomal subunits with 0.5 M LiCl was found to remove all but six of the ribosomal proteins. The proteins remaining associated with the (26S + 5.8S) rRNA complex were identified as L4, L8, L10, L12, L16 and L25. These core proteins were split off sequentially in the order (L16 + L12), L10, (L4 + L8), L25 by further increasing the LiCl concentration. At 1.0 M LiCl only ribosomal protein L25 remains bound to the rRNA. Upon lowering the LiCl concentration the core proteins reassociate with the rRNA in the reverse order of their removal. The susceptibility of the ribosomal proteins to removal by LiCl corresponds quite well with their order of assembly into the 60S subunit in vivo as determined earlier [Kruiswijk et al. (1978) Biochim. Biophys. Acta 517, 378-389]. Binding studies in vitro using partially purified L25 showed that this protein binds specifically to 26S rRNA. Therefore our experiments for the first time directly identify a eukaryotic ribosomal protein capable of binding to high-molecular-mass rRNA. Binding studies in vitro using a blot technique demonstrated that core proteins L8 and L16 as well as protein L21, though not present in any of the core particles, are also capable of binding to 26S rRNA to approximately the same extent as L25. About nine additional 60S proteins appeared to interact with the 26S rRNA, though to a lesser extent.     

Proteome phenotyping of acid stress-resistant mutants of Lactococcus lactis MG1363

To cope with medium acidity, Lactococcus lactis has evolved a number of inducible mechanisms commonly referred as acid stress response. To better understand the molecular basis of this response, several mutants constitutively tolerant to acidity were previously obtained by insertional random mutagenesis of L. lactis MG1363. Mutants in which the GMP synthase gene (i.e. guaA), the (p)ppGpp synthase gene (i.e. relA*) or the high affinity phosphate transport system (i.e. pstS) are inactivated are further characterized in this study. 2-DE was performed and showed that 42, 26, and 35 protein spots are positively deregulated in the guaA, relA*, and pstS mutants, respectively, as compared to the wild-type strain. Most of these proteins were identified by MS. Proteomes comparison of the mutants guaA, relA*, and pstS as well as the acid adaptation proteome of the wild-type strain revealed (i) the presence of numerous overlaps and (ii) that only five proteins were overexpressed in the four conditions, suggesting that these proteins play a crucial role in the constitutive acid stress tolerance of the mutants and in the acid tolerance response of the wild-type strain.     

Potential for cross-transmission of Dictyocaulus viviparus between cattle and white-tailed deer

Development of an in vitro culture system for infectious Dictyocaulus viviparus larvae made it possible to study the potential cross-transmission of D. viviparus between white-tailed deer (Odocoileus virginianus) and cattle (Bos taurus). Between 26 September 1995-29 February 1996, six parasite-free bull calves were individually inoculated with 15 to 50 infective third stage larvae (L3)/kg of body weight cultured from adult D. viviparus collected from white-tailed deer. Three bull calves were simultaneously inoculated with 45 L3/kg of body weight recovered from cattle either by the Baermann technique or by in vitro culture as above. All three calves inoculated with the homologous cattle strain became patently infected while all six calves inoculated with the heterologous deer strain remained negative for the presence of D. viviparus in the feces and in the lungs upon necropsy.     

The UBA domains of NUB1L are required for binding but not for accelerated degradation of the ubiquitin-like modifier FAT10

Proteins selected for degradation are labeled with multiple molecules of ubiquitin and are subsequently cleaved by the 26 S proteasome. A family of proteins containing at least one ubiquitin-associated (UBA) domain and one ubiquitin-like (UBL) domain have been shown to act as soluble ubiquitin receptors of the 26 S proteasome and introduce a new level of specificity into the degradation system. They bind ubiquitylated proteins via their UBA domains and the 26 S proteasome via their UBL domain and facilitate the contact between substrate and protease. NEDD8 ultimate buster-1 long (NUB1L) belongs to this class of proteins and contains one UBL and three UBA domains. We recently reported that NUB1L interacts with the ubiquitin-like modifier FAT10 and accelerates its degradation and that of its conjugates. Here we show that a deletion mutant of NUB1L lacking the UBL domain is still able to bind FAT10 but not the proteasome and no longer accelerates FAT10 degradation. A version of NUB1L lacking all three UBA domains, on the other hand, looses the ability to bind FAT10 but is still able to interact with the proteasome and accelerates the degradation of FAT10. The degradation of a FAT10 mutant containing only the C-terminal UBL domain is also still accelerated by NUB1L, even though the two proteins do not interact. In addition, we show that FAT10 and either one of its UBL domains alone can interact directly with the 26 S proteasome. We propose that NUB1L not only acts as a linker between the 26 S proteasome and ubiquitin-like proteins, but also as a facilitator of proteasomal degradation.     

Biodegradation of Maya crude oil fractions by bacterial strains and a defined mixed culture isolated from Cyperus laxus rhizosphere soil in a contaminated site

Ten bacterial strains were isolated by enrichment culture, using as carbon sources either aliphatics or an aromatic-polar mixture. Oxygen uptake rate was used as a criterion to determine culture transfer timing at each enrichment stage. Biodegradation of aliphatics (10,000 mg L(-1)) and an aromatic-polar mixture (5000 mg L(-1), 2:1) was evaluated for each of the bacterial strains and for a defined culture made up with a standardized mixture of the isolated strains. Degradation of total hydrocarbons (10,000 mg L(-1)) was also determined for the defined mixed culture. Five bacterial strains were able to degrade more than 50% of the aliphatic fraction. The most extensive biodegradation (74%) was obtained with strain Bs 9A, while strains Ps 2AP and UAM 10AP were able to degrade up to 15% of the aromatic-polar mixture. The defined mixed culture degraded 47% of the aliphatics and 6% of the aromatic-polar mixture. The defined mixed culture was able to degrade about 40% of the aliphatic fraction and 26% of the aromatic fraction when grown in the presence of total hydrocarbons, while these microorganisms did not consume the polar hydrocarbons fraction. The proposed strategy that combines enrichment culture together with oxygen uptake rate allowed the isolation of bacterial strains that are able to degrade specific hydrocarbons fractions at high consumption rates.     

Biotransformation of eugenol to vanillin by a novel strain Bacillus safensis SMS1003

Due to the extensive applications of vanillin as flavored compound and increasing consumers concern for its natural and environment friendly mode of production, present work was focused on the selection of bacterial isolate capable of producing vanillin using eugenol biotransformation. Bacterial strain SMS1003 is evidenced as the potential strain for vanillin production and identified as Bacillus safensis (GeneBank accession no. MG561863) using biochemical tests and molecular phylogenic analysis of its 16S rDNA gene sequence. Molar yield of vanillin reached up to 10.7% (0.055?g/L) at 96?h of biotransformation using growing culture of B. safensis SMS1003 in following culture conditions: eugenol concentration 500?mg/L; temperature 37?°C; initial pH 7.0; inoculum volume 4%; volume of culture media 10%; and shaking speed 180?rpm. Vanillin was detected as the single metabolite with a molar yield of 26% (0.12?g/L) at 96?h using resting cells of B. safensis SMS1003. Product confirmation was based on spectral scan using photodiode array detector, Fourier-transform infrared spectroscopy, high-performance liquid chromatography, and mass spectroscopy.     

Specificities of RNA N-glycosidase activity of Mirabilis antiviral protein variants.

Mirabilis antiviral protein (MAP), a ribosome-inactivating protein, inactivates both eukaryotic and prokaryotic ribosomes by means of site-specific RNA N-glycosidase activity. In order to identify the site of this activity, some amino acid residues of MAP, conserved in homologous ribosome-inactivating proteins, were altered to other amino acids by replacing DNA fragments of the total synthetic gene of MAP. When the in vitro proteins synthesis of rabbit reticulocyte was treated with MAP variants secreted into culture media of Escherichia coli transformants, the inhibitory effect of R26L and R48L (R26L designates MAP variant with Arg-26 changed to Leu) was found to be similar to that of native MAP. Both purified Y72F and Y118F had the same effect as native MAP, and E168D had a slightly weaker effect. In contrast, on the protein synthesis of E. coli, Y118F had one-tenth the effect of native MAP, and Y72F and E168D approximately one-hundredth the effect. These three variant proteins also exhibited reduced RNA N-glycosidase activity on substrate E. coli ribosomes. These results suggest that Tyr-72 and Glu-168 are involved in RNA N-glycosidase activity. When the R171K gene was expressed in E. coli, an N-glycosidic bond of the 23 S rRNA of the host ribosome was found to be cleaved, although no product of the gene could be detected. This suggests that MAP variants can maintain their N-glycosidase activity when the conserved Glu-168 and Arg-171 are changed to similarly charged residues.     

一株瘤胃源乳酸利用菌的分离鉴定及其体外代谢特性

【目的】从饲喂高精料的本地山羊瘤胃内分离到一株利用乳酸并能产生大量丙酸的菌株L9,并进一步研究了该菌在调控瘤胃微生物发酵中的作用。【方法】采用厌氧培养技术,结合形态、生理生化特性和16SrRNA基因序列分析结果。【结果】该菌株被鉴定为反刍兽新月形单胞菌（Selenomonas ruminantium）。该菌株体外代谢特性研究表明,L9可利用乳酸作为唯一碳源,该菌在24h内可对90mmol／L的乳酸完全降解。体外摸拟瘤胃急性酸中毒的发酵试验结果表明,以淀粉为底物时,与对照组相比,添加菌株L9可显著降低瘤胃微生物体外培养体系中乳酸浓度,提高pH值,提高总挥发性脂肪酸和丙酸浓度,并显著降低乙酸与丙酸的浓度比（P〈0．05）。【结论】结果显示,菌株L9是一株可代谢乳酸,促进丙酸生成,提高总挥发性脂肪酸浓度的有益瘤胃细菌。     

Ultrastructural study of the TG180 murine sarcoma cell invasion by Toxoplasma gondii: comparison between in vivo and in vitro cell cultures

Infection of non-adherent TG180 murine sarcoma cells with Toxoplasma gondii was compared, at the ultrastructural level, in both in vivo and in vitro conditions. Suspensions of 3.0 x 10(6) TG180 cells infected in vitro with 1.0 x 10(6) parasites of the RH strain were harvested between the first and 6th day post-infection and processed for transmission electron microscopy. In vivo infection was made by intraperitoneal inoculation in mice of 1.0 x 10(6) TG180 cells, that were co-inoculated with a parasite suspension at the same cell concentration. Cells were harvested 10, 20, 30 min and 24, 48 h post-inoculation and processed for transmission electron microscopy at the same conditions of the in vitro culture. It was observed TG180 murine sarcoma cells with intense and equivalent intracellular parasitism in both conditions. Host cells with parasitophorous vacuoles containing up to 16 parasites, as well as parasites undergoing mitoses or presenting a bradyzoite-like morphology, were frequently seen in both culture methods.     

一种增加毕赤酵母生产胰岛素前体的方法

为了提高胰岛素前体(PI)的产量,构建了p PIC9K-PI表达载体并电转化至毕赤酵母菌株GS115中,在浓度为4.0 mg/m L的G418抗性平板上筛选到了1株拷贝数为12的菌株CL012。将SNAREs(可溶性N-乙基马来酰亚胺敏感因子受体蛋白)组分中的SNC2和SNC2-SSO2分别转入菌株CL012中,并在摇瓶和5 L发酵罐水平上检测SNAREs对PI产量的影响。结果表明:摇瓶水平上,甲醇诱导96 h后,菌株CL012的PI产量为1.53 mg/L;表达SNC2和SNC2-SSO2的菌株的PI产量分别为1.89 mg/L和2.21 mg/L,分别比菌株CL012提高了23.53%和44.44%。在5 L发酵罐上进行高密度发酵,甲醇诱导96 h后菌株CL012的PI产量为53 mg/L,是摇瓶水平的34.64倍;表达SNC2和SNC2-SSO2的菌株的PI产量分别达到64 mg/L和78 mg/L,分别比菌株CL012提高了20.75%和47.17%。由此得出结论 SNAREs可以促进胰岛素前体的分泌,从而提高在毕赤酵母中的异源表达。     

Veneza zonata (Hemiptera: Coreidae)/trypanosomatid relationship: action of hemolymph in vitro and experimental infection

The defense response of Veneza zonata (Hemiptera: Coreidae) against three different trypanosomatid infections was assessed: (1) strain 714TD, a Leptomonas which has V. zonata as vector of a plant trypanosomatid, (2) strain 563TD, a Leptomonas isolated from the digestive tract of Euchistus heros (Hemiptera: Pentatomidae), and (3) Leishmania (L.) amazonensis, a human parasite that cannot infect V. zonata. Experiments with V. zonata hemolymph showed agglutination only of L. (L.) amazonensis culture forms and hemocytic recognition was more intense with this strain. L. (L.) amazonensis also activated the prophenoloxidase system, whereas strains 714TD and 563TD did not activate this system but rather seemed to inhibit phenoloxidase activity. No flagellates were seen in the digestive tract, hemolymph, or salivary glands in insects infected with L. (L.) amazonensis. The digestive tract, the hemolymph, and the salivary glands of insects fed on tomatoes inoculated with 714TD are sequentially invaded by the flagellate, which is inoculated in plants together with saliva. Insects fed on tomatoes inoculated with 563TD exhibited culture forms in the digestive tract (6 days after) and hemocoel (three additional days); however, they died 12 to 14 days after exposure. The salivary glands in insects inoculated in the hemocoel with 714TD strain are rapidly invaded, whereas those with 563TD culture forms died approximately 24 h after infection. Bacterial proliferation in the hemocoel and hemocyte surface blebbing were seen in insects infected only with 563TD strain as the probable pathogenic mechanism of insect death.     

Paromomycin affects translation and vesicle-mediated trafficking as revealed by proteomics of paromomycin -susceptible -resistant Leishmania donovani

Leishmania donovani is a protozoan parasite that causes visceral leishmaniasis (VL) and is responsible for significant mortality and morbidity. Increasing resistance towards antimonial drugs poses a great challenge in chemotherapy of VL. Paromomycin is an aminoglycosidic antibiotic and is one of the drugs currently being used in the chemotherapy of cutaneous and visceral leishmaniasis. To understand the mode of action of this antibiotic at the molecular level, we have investigated the global proteome differences between the wild type AG83 strain and a paromomycin resistant (PRr) strain of L. donovani. Stable isotope labeling of amino acids in cell culture (SILAC) followed by quantitative mass spectrometry of the wild type AG83 strain and the paromomycin resistant (PRr) strain identified a total of 226 proteins at ≥ 95% confidence. Data analysis revealed upregulation of 29 proteins and down-regulation of 21 proteins in the PRr strain. Comparative proteomic analysis of the wild type and the paromomycin resistant strains showed upregulation of the ribosomal proteins in the resistant strain indicating role in translation. Elevated levels of glycolytic enzymes and stress proteins were also observed in the PRr strain. Most importantly, we observed upregulation of proteins that may have a role in intracellular survival and vesicular trafficking in the PRr strain. Furthermore, ultra-structural analysis by electron microscopy demonstrated increased number of vesicular vacuoles in PRr strain when compared to the wild-type strain. Drug affinity pull-down assay followed by mass spectrometery identified proteins in L. donovani wild type strain that were specifically and covalently bound to paromomycin. These results provide the first comprehensive insight into the mode of action and underlying mechanism of resistance to paromomycin in Leishmania donovani.     

Vaccinia mature virus fusion regulator A26 protein binds to A16 and G9 proteins of the viral entry fusion complex and dissociates from mature virions at low pH

Vaccinia mature virus enters cells through either endocytosis or plasma membrane fusion, depending on virus strain and cell type. Our previous results showed that vaccinia virus mature virions containing viral A26 protein enter HeLa cells preferentially through endocytosis, whereas mature virions lacking A26 protein enter through plasma membrane fusion, leading us to propose that A26 acts as an acid-sensitive fusion suppressor for mature virus (S. J. Chang, Y. X. Chang, R. Izmailyan R, Y. L. Tang, and W. Chang, J. Virol. 84:8422-8432, 2010). In the present study, we investigated the fusion suppression mechanism of A26 protein. We found that A26 protein was coimmunoprecipitated with multiple components of the viral entry-fusion complex (EFC) in infected HeLa cells. Transient expression of viral EFC components in HeLa cells revealed that vaccinia virus A26 protein interacted directly with A16 and G9 but not with G3, L5 and H2 proteins of the EFC components. Consistently, a glutathione S-transferase (GST)-A26 fusion protein, but not GST, pulled down A16 and G9 proteins individually in vitro. Together, our results supported the idea that A26 protein binds to A16 and G9 protein at neutral pH contributing to suppression of vaccinia virus-triggered membrane fusion from without. Since vaccinia virus extracellular envelope proteins A56/K2 were recently shown to bind to the A16/G9 subcomplex to suppress virus-induced fusion from within, our results also highlight an evolutionary convergence in which vaccinia viral fusion suppressor proteins regulate membrane fusion by targeting the A16 and G9 components of the viral EFC complex. Finally, we provide evidence that acid (pH 4.7) treatment induced A26 protein and A26-A27 protein complexes of 70 kDa and 90 kDa to dissociate from mature virions, suggesting that the structure of A26 protein is acid sensitive.